Chloride channels are found in the plasma membranes of virtually every cell in the body. Chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ion across epithelial membranes. When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also regulate organelle pH (cf. Greger, R. (1988) Annu. Rev. Physiol. 50:111-122). Electrophysiological and pharmacological measurements including ion conductance, current-voltage relationships, and sensitivity to modulators suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
Several chloride channels have been cloned from mammalian tissues and cell lines. The sequences of these proteins are diverse and include a nicotinic acetylcholine receptor homolog, the cystic fibrosis transmembrane conductance regulator, and the highly acidic p64 protein with no significant homology to other proteins (Bernard, E. A. et al. (1987) Trends Neurosci. 16:502-509; Riordan, J. R. et al. (1989) Science 245:1066-1073; Landry, D. et al. (1993) J. Biol. Chem. 268:14948-14955). Many of the channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II which regulate chloride channel activity in epithelial cells.
The p64 protein was originally identified in bovine kidney cortex membranes by its affinity for indanyloxyacetic acid, a known inhibitor of epithelial chloride channels (Landry et al. (1989) Science 244:1469-1472). Antibodies raised against the isolated p64 protein can deplete solubilized kidney membranes of all detectable chloride channel activity. Thus, p64 is likely to be a functional component of the kidney chloride channel (Redhead, R. C. et al. (1992) Proc. Natl. Acad. Sci. 89:3716-3720).
Northern blot analyses using the p64 clone as a probe detect related mRNAs, ranging in size from .about.2 kb to .about.6.5 kb, in bovine kidney cortex, kidney medulla, liver, adrenal glands, brain, skeletal muscle, and heart. Most of these tissues have multiple transcripts that are capable of hybridizing to this probe. The diversity and relative abundance of these transcripts is tissue-specific, and this suggests that the p64 transcripts are alternatively spliced and/or that a family of related genes are expressed (Landry et al., supra).
The sequence of p64 predicts an acidic, integral membrane protein which spans the membrane at least twice and has the amino terminus on the cytoplasmic side. The protein has potential sites for phosphorylation by protein kinase C, tyrosine kinase, and casein kinase II, and a single site for N-linked glycosylation at Asp.sup.235.
The discovery of polynucleotides encoding a novel human anion channel, and the molecules themselves, provides a means to investigate the regulation of membrane potentials, intracellular pH, cell volume, signal transduction, and transepithelial ion transport in tissues containing absorptive or secretory epithelia under normal and disease conditions. Discovery of a novel anion channel satisfies a need in the art by providing new compositions useful in diagnosing and treating cancer and developmental disorders.